The development of new drugs is still being actively pursued all over the world, but it is difficult to conclude that new drugs could be efficiently developed as compared to the efforts involved in the development. This is believed to be due to yet insufficient understanding of biomolecular functions. In fact, even the currently available drugs remain yet unclear, in many cases, about how they act upon the body to exhibit their pharmaceutical effects. For this reason, it is required for the development of new drugs to synthesize a number of candidate compounds. Accordingly, if the details of biomolecular mechanism involved in diseases can be clarified, the development of new drugs will be greatly streamlined and such is expected to contribute to the development of a groundbreaking molecular target drug. Recently, trace analysis has become required for the same subject in animal experiments with a new drug. It has thus been strongly desired to develop a noninvasive in vivo imaging technique also from moral and ethical aspects of laboratory animals.
In recent years, as represented by green fluorescence protein (GFP), the imaging technology that visualizes molecules involved in life phenomena has been playing an important role in the medical and biological fields as an innovative technique for elucidating biomolecular functions. On the other hand, techniques for imaging the desired molecules in living animals (in vivo molecular imaging) are still less developed and, above all, it has been actively investigated to develop molecular probes available for fluorescent or luminescent imaging in the near-infrared region (wavelength of 700 nm or longer) which is able to permeate through the living tissues (K. Kiyose et al., Chem. Asian J 2008, 3, 506).
The present inventors thought that if a practical near-infrared bioluminescence system is newly developed, a breakthrough in vivo imaging technology would be provided and have continued studies, focusing anew on coelenterazine (CTZ, 1) which is a typical bioluminescence substrate from a long time ago.
CTZ is commonly used as a luminescence substrate in many marine organisms, including photoprotein aequorin form jellyfish, Renilla luciferase from sea pansy, Gaussia luciferase from copepoda, Oplophorus luciferase from decapoda, etc. Accordingly, if a near-infrared luminescent CTZ compound (CTZ and its analogues) available for any of luciferases can be developed by modifying the structure of CTZ, it is expected that a luminescence imaging method applicable also to living animals could be provided for the detection with high sensitivity of in vivo localization of a target protein and its absolute quantity and metabolic rate, a promoter or enhancer activity of the target protein, life phenomena of a target cell accompanied by in vivo localization, etc.
Approximately 50 kinds of CTZ compounds have been synthesized so far. Some of them were examined for their substrate specificity in several bioluminescence systems.
Among them, v-coelenterazine (v-CTZ, 2) first reported by Shimomura, Kishi, et al. (O, Shimomura, Y. Kishi, et al., Biochem. J. 1988, 251, 405) was later studied for the luminescence properties of aequorin, Renilla luciferase and Oplophorus luciferase by Inouye and Shimomura. As a result, it was found that when used as a substrate for Renilla luciferase, the maximum emission wavelength was shifted toward the longer wavelength side, i.e., from 475 nm (blue emission) to 512 nm (green emission) by about 40 nm (S. Inouye & O, Shimomura, Biochem. Biophys. Res. Commun. 1997, 233, 349). It was also revealed that a part (˜5%) of this emission spectrum distribution reached the near-infrared region (>700 nm).

In addition, A. M. Loenig et al. recently achieved a longer wavelength shift by modifying the amino acid sequence of Renilla luciferase (A. M. Loenig et at, Nature Methods 2007, 4, 641). More specifically, they reported that the maximum emission wavelength was shifted to 547 nm (green emission) when v-CTZ was used as a substrate for the modified Renilla luciferase. In fact, they also succeeded in imaging in living mice by utilizing this luminescence system.
In order to establish a practical in vivo imaging system, however, it is desired to obtain an emission peak at a longer wavelength region. It is further pointed out that v-CTZ is easily oxidized and a problem also arises in stability (G. Stepanyuk et al., Anal. Bioanal. Chem. 2010, 398, 1809). It is therefore an important task to create a novel substrate practically applicable to the CTZ near-infrared bioluminescence system.
Furthermore, a process for producing v-CTZ has not been hitherto reported in detail, and it is also desired to establish the process.